Prions in skeletal muscles of deer with chronic wasting disease

The emergence of chronic wasting disease (CWD) in deer and elk in an increasingly wide geographic area, as well as the interspecies transmission of bovine spongiform encephalopathy to humans in the form of variant Creutzfeldt Jakob disease, have raised concerns about the zoonotic potential of CWD. Because meat consumption is the most likely means of exposure, it is important to determine whether skeletal muscle of diseased cervids contains prion infectivity. Here bioassays in transgenic mice expressing cervid prion protein revealed the presence of infectious prions in skeletal muscles of CWD-infected deer, demonstrating that humans consuming or handling meat from CWD-infected deer are at risk to prion exposure.     

Facilitated cross-species transmission of prions in extraneural tissue

Prions are infectious pathogens essentially composed of PrP(Sc), an abnormally folded form of the host-encoded prion protein PrP(C). Constrained steric interactions between PrP(Sc) and PrP(C) are thought to provide prions with species specificity and to control cross-species transmission into other host populations, including humans. We compared the ability of brain and lymphoid tissues from ovine and human PrP transgenic mice to replicate foreign, inefficiently transmitted prions. Lymphoid tissue was consistently more permissive than the brain to prions such as those causing chronic wasting disease and bovine spongiform encephalopathy. Furthermore, when the transmission barrier was overcome through strain shifting in the brain, a distinct agent propagated in the spleen, which retained the ability to infect the original host. Thus, prion cross-species transmission efficacy can exhibit a marked tissue dependence.     

Human prion protein with valine 129 prevents expression of variant CJD phenotype

Variant Creutzfeldt-Jakob disease (vCJD) is a unique and highly distinctive clinicopathological and molecular phenotype of human prion disease associated with infection with bovine spongiform encephalopathy (BSE)-like prions. Here, we found that generation of this phenotype in transgenic mice required expression of human prion protein (PrP) with methionine 129. Expression of human PrP with valine 129 resulted in a distinct phenotype and, remarkably, persistence of a barrier to transmission of BSE-derived prions on subpassage. Polymorphic residue 129 of human PrP dictated propagation of distinct prion strains after BSE prion infection. Thus, primary and secondary human infection with BSE-derived prions may result in sporadic CJD-like or novel phenotypes in addition to vCJD, depending on the genotype of the prion source and the recipient.     

Prevention of scrapie pathogenesis by transgenic expression of anti-prion protein antibodies

Variant Creutzfeldt-Jakob disease and bovine spongiform encephalopathy are initiated by extracerebral exposure to prions. Although prion transmission from extracerebral sites to the brain represents a potential target for prophylaxis, attempts at vaccination have been limited by the poor immunogenicity of prion proteins. To circumvent this, we expressed an anti-prion protein (anti-PrP) mu chain in Prnp(o/o) mice. Transgenic mice developed sustained anti-PrP titers, which were not suppressed by introduction of Prnp+ alleles. Transgene expression prevented pathogenesis of prions introduced by intraperitoneal injection in the spleen and brain. Expression of endogenous PrP (PrP(C)) in the spleen and brain was unaffected, suggesting that immunity was responsible for protection. This indicates the feasibility of immunological inhibition of prion disease in vivo.     

Chronic lymphocytic inflammation specifies the organ tropism of prions

Prions typically accumulate in nervous and lymphoid tissues. Because proinflammatory cytokines and immune cells are required for lymphoid prion replication, we tested whether inflammatory conditions affect prion pathogenesis. We administered prions to mice with five inflammatory diseases of the kidney, pancreas, or liver. In all cases, chronic lymphocytic inflammation enabled prion accumulation in otherwise prion-free organs. Inflammatory foci consistently correlated with lymphotoxin up-regulation and ectopic induction of FDC-M1+ cells expressing the normal cellular prion protein PrPC. By contrast, inflamed organs of mice lacking lymphotoxin-alpha or its receptor did not accumulate the abnormal isoform PrPSc, nor did they display infectivity upon prion inoculation. By expanding the tissue distribution of prions, chronic inflammatory conditions may act as modifiers of natural and iatrogenic prion transmission.     

Coincident scrapie infection and nephritis lead to urinary prion excretion

Prion infectivity is typically restricted to the central nervous and lymphatic systems of infected hosts, but chronic inflammation can expand the distribution of prions. We tested whether chronic inflammatory kidney disorders would trigger excretion of prion infectivity into urine. Urinary proteins from scrapie-infected mice with lymphocytic nephritis induced scrapie upon inoculation into noninfected indicator mice. Prionuria was found in presymptomatic scrapie-infected and in sick mice, whereas neither prionuria nor urinary PrP(Sc) was detectable in prion-infected wild-type or PrP(C)-overexpressing mice, or in nephritic mice inoculated with noninfectious brain. Thus, urine may provide a vector for horizontal prion transmission, and inflammation of excretory organs may influence prion spread.     

A general model of prion strains and their pathogenicity

Prions are lethal mammalian pathogens composed of aggregated conformational isomers of a host-encoded glycoprotein and which appear to lack nucleic acids. Their unique biology, allied with the public-health risks posed by prion zoonoses such as bovine spongiform encephalopathy, has focused much attention on the molecular basis of prion propagation and the "species barrier" that controls cross-species transmission. Both are intimately linked to understanding how multiple prion "strains" are encoded by a protein-only agent. The underlying mechanisms are clearly of much wider importance, and analogous protein-based inheritance mechanisms are recognized in yeast and fungi. Recent advances suggest that prions themselves are not directly neurotoxic, but rather their propagation involves production of toxic species, which may be uncoupled from infectivity.     

Creating a protein-based element of inheritance

Proteins capable of self-perpetuating changes in conformation and function (known as prions) can serve as genetic elements. To test whether novel prions could be created by recombinant methods, a yeast prion determinant was fused to the rat glucocorticoid receptor. The fusion protein existed in different heritable functional states, switched between states at a low spontaneous rate, and could be induced to switch by experimental manipulations. The complete change in phenotype achieved by transferring a prion determinant from one protein to another confirms the protein-only nature of prion inheritance and establishes a mechanism for engineering heritable changes in phenotype that should be broadly applicable.     

鹿慢性消耗性疾病免疫组织化学检测方法的建立与优化

【目的】在自主研制的抗鹿朊蛋白单克隆抗体基础上,建立和优化了鹿慢性消耗性疾病(chronic wasting disease, CWD)的免疫组织化学（immunohistochemistry, IHC）诊断方法。【方法】按照常规石蜡病理切片制作方法制备阴性和阳性切片,应用ABC法对获得的5株单抗进行免疫组织化学检测。【结果】试验结果表明,5株单抗（编号分别为5A5、3B2、6D12、5E3、1F5）中只有5E3和3B2可以与CWD的阳性鹿延髓切片发生特异性免疫反应,而且5E3的反应较强烈,3B2的反应较弱,其它3株单抗则在试验的浓度范围内无特异性免疫反应。此外,5E3的稀释比例为1：10 000时,免疫反应效果达到最佳状态。【结论】实验结果表明,单抗5E3可以用于我国CWD的监测。     

Synthetic mammalian prions

Recombinant mouse prion protein (recMoPrP) produced in Escherichia coli was polymerized into amyloid fibrils that represent a subset of beta sheet-rich structures. Fibrils consisting of recMoPrP(89-230) were inoculated intracerebrally into transgenic (Tg) mice expressing MoPrP(89-231). The mice developed neurologic dysfunction between 380 and 660 days after inoculation. Brain extracts showed protease-resistant PrP by Western blotting; these extracts transmitted disease to wild-type FVB mice and Tg mice overexpressing PrP, with incubation times of 150 and 90 days, respectively. Neuropathological findings suggest that a novel prion strain was created. Our results provide compelling evidence that prions are infectious proteins.     

Impaired prion replication in spleens of mice lacking functional follicular dendritic cells

In scrapie-infected mice, prions are found associated with splenic but not circulating B and T lymphocytes and in the stroma, which contains follicular dendritic cells (FDCs). Formation and maintenance of mature FDCs require the presence of B cells expressing membrane-bound lymphotoxin-alpha/beta. Treatment of mice with soluble lymphotoxin-beta receptor results in the disappearance of mature FDCs from the spleen. We show that this treatment abolishes splenic prion accumulation and retards neuroinvasion after intraperitoneal scrapie inoculation. These data provide evidence that FDCs are the principal sites for prion replication in the spleen.     

GAMMA-GLOBULINS: QUANTITATIVE RELATIONSHIPS IN HUMAN SERUM AND NONVASCULAR FLUIDS

Three types of gamma-globulins, gamma(2), gamma(IA), and yim, are present in certain body fluids and secretions in proportions significantly different from those of normal human serum. A lthough ylA-globulin is present in only small amounts in serum, it represents a major fraction of the gamma globulin of tears, bile, saliva, colostrum, and fluid of the small intestine.     

Prion-induced amyloid heart disease with high blood infectivity in transgenic mice

We investigated extraneural manifestations in scrapie-infected transgenic mice expressing prion protein lacking the glycophosphatydylinositol membrane anchor. In the brain, blood, and heart, both abnormal protease-resistant prion protein (PrPres) and prion infectivity were readily detected by immunoblot and by inoculation into nontransgenic recipients. The titer of infectious scrapie in blood plasma exceeded 10(7) 50% infectious doses per milliliter. The hearts of these transgenic mice contained PrPres-positive amyloid deposits that led to myocardial stiffness and cardiac disease.     

热力学因素对绵羊重组朊蛋白体外构象转化的作用

构建了绵羊朊蛋白的原核表达载体,获得了高纯度的融合表达蛋白。并在热力学因素的作用下,研究了重组绵羊朊蛋白的构象转化。以基因型为ARQ/ARQ蒙古绵羊的血液DNA为模板,利用DNA重组技术,将绵羊朊蛋白正常成熟蛋白基因OvPrP插入表达载体pET30a,在大肠杆菌BL2l(DE3)中高效表达,获得的表达产物以包涵体形式存在,并对其进行纯化和复性。超滤浓缩后浓度约为0.5 mg/mL的OvPrP97-234,进行热力学处理,利用远紫外线圆二色谱(CD)分析热力学处理前、后蛋白的二级结构的变化,同时,对热力学处理前、后的蛋白进行了蛋白酶K抗性的检测,并对其高级结构进行了预测。结果表明:获得的表达产物经SDS-PAGE分析可见分子量为16kD的蛋白条带,Western-blotting的鉴定证实了所获得的蛋白是特异性的朊蛋白。经Jascow32软件分析,测得天然构象的OvPrP97-234的二级结构含量为:α螺旋为28.8%、β折叠为0%、转角和无规卷曲为71.1%,无蛋白酶K的抗性。经过热力学处理之后,OvPrP97-234的二级结构含量为:α螺旋为19.3%,β折叠为36.9%,转角和无规卷曲为43.8%,有一定...     

Presymptomatic detection of prions in blood

Prions are thought to be the proteinaceous infectious agents responsible for transmissible spongiform encephalopathies (TSEs). PrP(Sc), the main component of the infectious agent, is also the only validated surrogate marker for the disease, and its sensitive detection is critical for minimizing the spread of the disease. We detected PrP(Sc) biochemically in the blood of hamsters infected with scrapie during most of the presymptomatic phase of the disease. At early stages of the incubation period, PrP(Sc) detected in blood was likely to be from the peripheral replication of prions, whereas at the symptomatic phase, PrP(Sc) in blood was more likely to have leaked from the brain. The ability to detect prions biochemically in the blood of infected but not clinically sick animals offers a great promise for the noninvasive early diagnosis of TSEs.     

冰灾后粤北常绿阔叶林粗死木质残体的组成与结构

应用样方调查法,研究冰灾后粤北常绿阔叶林粗死木质残体（coarse woody debris,CWD）的组成和结构特征,比较不同坡向林分的CWD存在形式、种类组成、径级结构、样方分布以及腐烂等级状态。结果表明：冰灾后迎风坡林分的CWD贮量为14.81 t/hm2,明显高于背风坡林分5.66 t/hm2的CWD贮量;且不同坡向林分的CWD存在形式有较大的差异,迎风坡林分中CWD存在形式以倒木为主,而背风坡林分则以枯立木为主;倒木的分解速度较快,主要处于中度分解状态,而大多数枯立木则处于轻度分解状态;迎风坡林分大径级树种由于积雪作用,形成大量倒木;而背风坡林分小径级树种受低温冻害的影响,大量枯萎死亡。冰灾引起的CWD剧增会严重影响森林演替的进程,其中倒木对森林生态系统结构和功能短期的调节作用比较明显,而枯立木对森林生态系统的影响是一个长期的过程,有必要对其进行长期量化研究。     

狍被毛髓质指数在保温和保护功能上的适应性变化

应用显微测微尺，对狍肩部和前肢部的和立夏被毛进行髓质指数测算，差异显著性检验表明，狍被毛的髓质指数既显著的季节性差异。也存在身体部位差异，即躯干部冬毛的髓质变得极为发达、肢体部冬毛的髓质却显著变细。     

天宝岩典型森林群落粗死木质残体现存量研究

对天宝岩国家级自然保护区长苞铁杉林、猴头杜鹃林和柳杉林典型森林群落粗死木质残体（CWD）现存量进行研究。结果表明:1）天宝岩3种典型森林群落CWD现存量分别为33.6、45.3、31.8 t/hm2,处于我国热带和亚热带地区森林的中间水平;2）3种森林群落中,CWD的不同形态组成较不同腐烂等级对现存量差异的贡献度更大;3）猴头杜鹃林和长苞铁杉林内各种类型CWD现存量表现为倒木枯立木树桩,柳杉林内CWD现存量则为树桩枯立木倒木;4）中高腐烂等级CWD现存量为猴头杜鹃林长苞铁杉林柳杉林,而在低腐烂等级中呈现长苞铁杉林猴头杜鹃林;5）在柳杉林内,未受人为干扰地段枯立木和树桩所占比例明显高于受干扰地段,而倒木的比例则很低。      

不同鹿茸片红外数据的聚类分析

[目的]探讨不同鹿茸片的有效鉴别方法。[方法]采用SPSS软件对梅花鹿茸片、去血梅花鹿茸片、劣等梅花鹿茸片、假鹿茸片的红外光谱数据进行聚类分析。[结果]4种鹿茸片的红外光谱图谱存在明显差异;聚类分析表明,去血梅花鹿茸片和梅花鹿茸片为一类,成分没有明显差异。[结论]傅里叶变换红外光谱（FT-IR）和社会科学统计软件包（SPSS）软件相结合为梅花鹿茸的质量鉴别提供了一个客观有效的方法。     

森林粗木质残体研究进展

森林粗木质残体是指森林中林木生长的竞争排斥、老龄林内树木自然死亡、自然干扰(风倒、火灾、雨淋、雪折、病虫害、泥石流灾害、真菌侵害等)以及人为干扰(伐木、砍樵)等而形成。从粗木质残体的概念及其类型,以及森林生态系统中生产者、消费者、分解者的营养结构等方面综述了粗木质残体的功能。根据国内外对粗木质残体的研究情况详细论述了其研究方法,并对我国森林粗木质残体的研究进行了展望。